Blocking IL-6 signaling improves glucose tolerance via SLC39A5-mediated suppression of glucagon secretion.

BACKGROUND AND AIMS: Hyperinsulinemia, hyperglucagonemia, and low-grade inflammation are frequently presented in obesity and type 2 diabetes (T2D). The pathogenic regulation between hyperinsulinemia/insulin resistance (IR) and low-grade inflammation is well documented in the development of diabetes. However, the cross-talk of hyperglucagonemia with low-grade inflammation during diabetes progression is poorly understood. In this study, we investigated the regulatory role of proinflammatory cytokine interleukin-6 (IL-6) on glucagon secretion. METHODS: The correlations between inflammatory cytokines and glucagon or insulin were analyzed in rhesus monkeys and humans. IL-6 signaling was blocked by IL-6 receptor-neutralizing antibody tocilizumab in obese or T2D rhesus monkeys, glucose tolerance was evaluated by intravenous glucose tolerance test (IVGTT). Glucagon and insulin secretion were measured in isolated islets from wild-type mouse, primary pancreatic α-cells and non-α-cells sorted from GluCre-ROSA26EYFP (GYY) mice, in which the enhanced yellow fluorescent protein (EYFP) was expressed under the proglucagon promoter, by fluorescence-activated cell sorting (FACS). Particularly, glucagon secretion in α-TC1 cells treated with IL-6 was measured, and RNA sequencing was used to screen the mediator underlying IL-6-induced glucagon secretion. SLC39A5 was knocking-down or overexpressed in α-TC1 cells to determine its impact in glucagon secretion and cytosolic zinc density. Dual luciferase and chromatin Immunoprecipitation were applied to analyze the signal transducer and activator of transcription 3 (STAT3) in the regulation of SLC39A5 transcription. RESULTS: Plasma IL-6 correlate positively with plasma glucagon levels, but not insulin, in rhesus monkeys and humans. Tocilizumab treatment reduced plasma glucagon, blood glucose and HbA1c in spontaneously obese or T2D rhesus monkeys. Tocilizumab treatment also decreased glucagon levels during IVGTT, and improved glucose tolerance. Moreover, IL-6 significantly increased glucagon secretion in isolated islets, primary pancreatic α-cells and α-TC1 cells. Mechanistically, we found that IL-6-activated STAT3 downregulated the zinc transporter SLC39A5, which in turn reduced cytosolic zinc concentration and ATP-sensitive potassium channel activity and augmented glucagon secretion. CONCLUSIONS: This study demonstrates that IL-6 increases glucagon secretion via the downregulation of zinc transporter SLC39A5. This result revealed the molecular mechanism underlying the pathogenesis of hyperglucagonemia and a previously unidentified function of IL-6 in the pathophysiology of T2D, providing a potential new therapeutic strategy of targeting IL-6/glucagon to preventing or treating T2D.

Targeting ANGPTL3 by GalNAc-conjugated siRNA ANGsiR10 lowers blood lipids with long-lasting and potent efficacy in mice and monkeys.

Angiopoietin-like protein 3 (ANGPTL3) is an important regulator of lipoproteins by inhibiting both lipoprotein and endothelial lipases. It has been intensively investigated as a drug target for the treatment of dyslipidemia. In the present study, a modified small interfering RNA (siRNA) conjugated with GalNAc ANGsiR10 was characterized by in vivo and in vitro studies for its effect on ANGPTL3 silencing, the reduction of plasma triglycerides (TGs), and cholesterol levels in disease models. The results showed that ANGsiR10 displayed a significant and long-lasting efficacy in reducing blood TG and cholesterol levels in both mice and monkeys. Remarkably, the maximal reductions of plasma TG levels in the hApoC3-Tg mice, a model with high TG levels, and the spontaneous dyslipidemia model of rhesus monkey were 96.3% and 67.7%, respectively, after a single dose of ANGsiR10, with long-lasting effects up to 15 weeks. The cholesterol levels were also reduced in response to treatment, especially the non-HDL-c level, without altering the ApoA/ApoB ratio. This study showed that ANGsiR10 is effective in treating dyslipidemia and is worth further development.

Dusp6 deficiency attenuates neutrophil-mediated cardiac damage in the acute inflammatory phase of myocardial infarction.

Dual-specificity phosphatase 6 (DUSP6) serves a specific and conserved function on the dephosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). We previously identified Dusp6 as a regenerative repressor during zebrafish heart regeneration, therefore we propose to investigate the role of this repressor in mammalian cardiac repair. Utilizing a rat strain harboring Dusp6 nonsense mutation, rat neutrophil-cardiomyocyte co-culture, bone marrow transplanted rats and neutrophil-specific Dusp6 knockout mice, we find that Dusp6 deficiency improves cardiac outcomes by predominantly attenuating neutrophil-mediated myocardial damage in acute inflammatory phase after myocardial infarction. Mechanistically, Dusp6 is transcriptionally activated by p38-C/EBPß signaling and acts as an effector for maintaining p-p38 activity by down-regulating pERK and p38-targeting phosphatases DUSP1/DUSP16. Our findings provide robust animal models and novel insights for neutrophil-mediated cardiac damage and demonstrate the potential of DUSP6 as a therapeutic target for post-MI cardiac remodeling and other relevant inflammatory diseases.

Novel CaMKII-δ Inhibitor Hesperadin Exerts Dual Functions to Ameliorate Cardiac Ischemia/Reperfusion Injury and Inhibit Tumor Growth.

BACKGROUND: Cardiac ischemia/reperfusion (I/R) injury has emerged as an important therapeutic target for ischemic heart disease, the leading cause of morbidity and mortality worldwide. At present, there is no effective therapy for reducing cardiac I/R injury. CaMKII (Ca2+/calmodulin-dependent kinase II) plays a pivotal role in the pathogenesis of severe heart conditions, including I/R injury. Pharmacological inhibition of CaMKII is an important strategy in the protection against myocardial damage and cardiac diseases. To date, there is no drug targeting CaMKII for the clinical therapy of heart disease. Furthermore, at present, there is no selective inhibitor of CaMKII-δ, the major CaMKII isoform in the heart. METHODS: A small-molecule kinase inhibitor library and a high-throughput screening system for the kinase activity assay of CaMKII-δ9 (the most abundant CaMKII-δ splice variant in human heart) were used to screen for CaMKII-δ inhibitors. Using cultured neonatal rat ventricular myocytes, human embryonic stem cell-derived cardiomyocytes, and in vivo mouse models, in conjunction with myocardial injury induced by I/R (or hypoxia/reoxygenation) and CaMKII-δ9 overexpression, we sought to investigate the protection of hesperadin against cardiomyocyte death and cardiac diseases. BALB/c nude mice with xenografted tumors of human cancer cells were used to evaluate the in vivo antitumor effect of hesperadin. RESULTS: Based on the small-molecule kinase inhibitor library and screening system, we found that hesperadin, an Aurora B kinase inhibitor with antitumor activity in vitro, directly bound to CaMKII-δ and specifically blocked its activation in an ATP-competitive manner. Hesperadin functionally ameliorated both I/R- and overexpressed CaMKII-δ9-induced cardiomyocyte death, myocardial damage, and heart failure in both rodents and human embryonic stem cell-derived cardiomyocytes. In addition, in an in vivo BALB/c nude mouse model with xenografted tumors of human cancer cells, hesperadin delayed tumor growth without inducing cardiomyocyte death or cardiac injury. CONCLUSIONS: Here, we identified hesperadin as a specific small-molecule inhibitor of CaMKII-δ with dual functions of cardioprotective and antitumor effects. These findings not only suggest that hesperadin is a promising leading compound for clinical therapy of cardiac I/R injury and heart failure, but also provide a strategy for the joint therapy of cancer and cardiovascular disease caused by anticancer treatment.

Targeting CaMKII-δ9 Ameliorates Cardiac Ischemia/Reperfusion Injury by Inhibiting Myocardial Inflammation.

BACKGROUND: CaMKII (Ca2+/calmodulin-dependent kinase II) plays a central role in cardiac ischemia/reperfusion (I/R) injury-an important therapeutic target for ischemic heart disease. In the heart, CaMKII-δ is the predominant isoform and further alternatively spliced into 11 variants. In humans, CaMKII-δ9 and CaMKII-δ3, the major cardiac splice variants, inversely regulate cardiomyocyte viability with the former pro-death and the latter pro-survival. However, it is unknown whether specific inhibition of the detrimental CaMKII-δ9 prevents cardiac I/R injury and, if so, what is the underlying mechanism. Here, we aim to investigate the cardioprotective effect of specific CaMKII-δ9 inhibition against myocardial I/R damage and determine the underlying mechanisms. METHODS: The role and mechanism of CaMKII-δ9 in cardiac I/R injury were investigated in mice in vivo, neonatal rat ventricular myocytes, and human embryonic stem cell-derived cardiomyocytes. RESULTS: We demonstrate that CaMKII-δ9 inhibition with knockdown or knockout of its feature exon, exon 16, protects the heart against I/R-elicited injury and subsequent heart failure. I/R-induced cardiac inflammation was also ameliorated by CaMKII-δ9 inhibition, and compared with the previously well-studied CaMKII-δ2, CaMKII-δ9 overexpression caused more profound cardiac inflammation. Mechanistically, in addition to IKKß (inhibitor of NF-κB [nuclear factor-κB] kinase subunit ß), CaMKII-δ9, but not δ2, directly interacted with IκBα (NF-κB inhibitor α) with its feature exon 13-16-17 combination and increased IκBα phosphorylation and consequently elicited more pronounced activation of NF-κB signaling and inflammatory response. Furthermore, the essential role of CaMKII-δ9 in myocardial inflammation and damage was confirmed in human cardiomyocytes. CONCLUSIONS: We not only identified CaMKII-δ9-IKK/IκB-NF-κB signaling as a new regulator of human cardiomyocyte inflammation but also demonstrated that specifically targeting CaMKII-δ9, the most abundant CaMKII-δ splice variant in human heart, markedly suppresses I/R-induced cardiac NF-κB activation, inflammation, and injury and subsequently ameliorates myocardial remodeling and heart failure, providing a novel therapeutic strategy for various ischemic heart diseases.

CaMKII-δ9 Induces Cardiomyocyte Death to Promote Cardiomyopathy and Heart Failure.

Heart failure is a syndrome in which the heart cannot pump enough blood to meet the body's needs, resulting from impaired ventricular filling or ejection of blood. Heart failure is still a global public health problem and remains a substantial unmet medical need. Therefore, it is crucial to identify new therapeutic targets for heart failure. Ca2+/calmodulin-dependent kinase II (CaMKII) is a serine/threonine protein kinase that modulates various cardiac diseases. CaMKII-δ9 is the most abundant CaMKII-δ splice variant in the human heart and acts as a central mediator of DNA damage and cell death in cardiomyocytes. Here, we proved that CaMKII-δ9 mediated cardiomyocyte death promotes cardiomyopathy and heart failure. However, CaMKII-δ9 did not directly regulate cardiac hypertrophy. Furthermore, we also showed that CaMKII-δ9 induced cell death in adult cardiomyocytes through impairing the UBE2T/DNA repair signaling. Finally, we demonstrated no gender difference in the expression of CaMKII-δ9 in the hearts, together with its related cardiac pathology. These findings deepen our understanding of the role of CaMKII-δ9 in cardiac pathology and provide new insights into the mechanisms and therapy of heart failure.

Dynamic changes in insulin and glucagon during disease progression in rhesus monkeys with obesity-related type 2 diabetes mellitus.

AIMS: To investigate the progression of obesity-related type 2 diabetes mellitus (T2DM) in rhesus monkeys, especially dynamic changes in insulin and glucagon. MATERIALS AND METHODS: We followed a cohort of 52 rhesus monkeys for 7 years throughout the progression of obesity-related T2DM. Intravenous glucose tolerance tests were performed every 6 months to evaluate dynamic changes in glucose, insulin and glucagon levels. RESULTS: Obesity in rhesus monkeys increased the overall mortality and T2DM morbidity. During the progression of T2DM, glucagon remained consistently elevated, while insulin initially increased in compensation but then dropped to below normal levels when the monkeys developed overt T2DM. After a glucose challenge, both the first and second phases of insulin secretion increased during the early stage of T2DM; in later stages the first phase was delayed and the second phase was diminished. CONCLUSION: Our findings showed that, beside the decreased insulin level, hyperglucagonaemia also plays an important role in the development of T2DM.

Central role of RIPK1-VDAC1 pathway on cardiac impairment in a non-human primate model of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disorder characterized by destructive polyarthritis and systemic complications. It increases cardiovascular morbidity and mortality. However, the mechanism underlying RA-related cardiac damage remains largely unknown. Here, we found and characterized a non-human primate (NHP) model with spontaneous RA similar to the human conditions. Compared with the control group, the cardiac function in RA monkeys showed progressively deterioration; histologically, we found significantly increased inflammatory cell infiltration, cell death, and fibrosis in RA monkey heart tissue. Mechanistically, the upregulated receptor-interacting protein kinase 1 (RIPK1) in RA monkey heart tissue bound to voltage-dependent anion-selective channel 1 (VDAC1), increased VDAC1 oligomerization, and subsequently induced cardiac cell death and functional impairment. These findings identified that RIPK1-VDAC1 pathway is a promising target to treat cardiac impairment in RA. This unique model of RA will provide a valuable tool for mechanistic and translational studies.

Characterization of spontaneously-developed non-alcoholic fatty liver disease in aged rhesus monkeys.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a global epidemic afflicting 20-30% in the general population. The animal model of NAFLD available at the present are less clinically relevant. In this study. We aimed to establish a NAFLD model of rhesus monkeys and develop an ultrasonographic steatosis score (USS) system to grade hepatic steatosis in this model. METHODS: We performed hepatic ultrasonography and blood biochemical tests on 86 rhesus monkeys with and without metabolic syndrome (MetS), among which 45 animals were further assessed by histopathological analysis. RESULTS: The liver histological features of rhesus monkeys NAFLD were resemble to those of NAFLD patients. There was a close correlation between the histological steatosis grade and the USS (Spearman's coefficient, 0.705, p < 0.001). The USS sensitivity was 87.5% and the specificity was 94.6% when the cut-off was USS2. In addition, the prevalence of MetS was significantly higher in the USS2-3 group. Multiple risk factors of cardiometabolic disease, including obesity, insulin resistance and dyslipidemia were significantly correlated with the USS. CONCLUSIONS: NAFLD was developed spontaneously among aging in rhesus monkeys (with increased prevalence in the MetS monkeys), which provided an ideal model for NAFLD. The newly developed USS system can be used to evaluate fatty liver in the rhesus monkey. The model as well as the noninvasive assessment methodology will provide a powerful tool for mechanistic studies and preclinical test of novel therapies for NAFLD.

Deficiency of PRKD2 triggers hyperinsulinemia and metabolic disorders.

Hyperinsulinemia is the earliest symptom of insulin resistance (IR), but a causal relationship between the two remains to be established. Here we show that a protein kinase D2 (PRKD2) nonsense mutation (K410X) in two rhesus monkeys with extreme hyperinsulinemia along with IR and metabolic defects by using extreme phenotype sampling and deep sequencing analyses. This mutation reduces PRKD2 at both the mRNA and the protein levels. Taking advantage of a PRKD2-KO mouse model, we demonstrate that PRKD2 deletion triggers hyperinsulinemia which precedes to IR and metabolic disorders in the PRKD2 ablation mice. PRKD2 deficiency promotes ß-cell insulin secretion by increasing the expression and activity of L-type Ca2+ channels and subsequently augmenting high glucose- and membrane depolarization-induced Ca2+ influx. Altogether, these results indicate that down-regulation of PRKD2 is involved in the pathogenesis of hyperinsulinemia which, in turn, results in IR and metabolic disorders.

Mitochondria Associated MicroRNA Expression Profiling of Heart Failure.

Heart failure (HF) is associated with mitochondrial dysfunction and energy metabolism impairment. MicroRNAs are implicated in the development of heart failure. However, the mitochondria enriched microRNA during heart failure remains elusive. Here, we generated a pressure overload-induced early and late stage heart failure model at 4 weeks and 8 weeks following transverse aortic constriction (TAC) in mice. We found that expression of mitochondrion protein COX4 was highly enriched in isolated mitochondria from cardiac tissues while GAPDH could hardly be detected. Furthermore, small RNA sequencing for mitochondria RNAs from failing hearts was performed. It was found that 69 microRNAs were upregulated and 2 were downregulated in early heart failure, while 16 microRNAs were upregulated and 6 were downregulated in late heart failure. 15 microRNA candidates were measured in both mitochondria and total cardiac tissues of heart failure by real-time PCR. MiR-696, miR-532, miR-690, and miR-345-3p were enriched in mitochondria from the failing heart at early stage. Bioinformatics analysis showed that mitochondria enriched microRNAs in HF were associated with energy metabolism and oxidative stress pathway. For the first time, we demonstrated microRNAs were enriched in mitochondria during heart failure, which established a link between microRNA and mitochondrion in heart failure.

Eplerenone restores 24-h blood pressure circadian rhythm and reduces advanced glycation end-products in rhesus macaques with spontaneous hypertensive metabolic syndrome.

Hypertension is often associated with metabolic syndrome (MetS), and serves as a risk factor of MetS and its complications. Blood pressure circadian rhythm in hypertensive patients has been suggested to contribute to cardiovascular consequences and organ damage of hypertension. But circadian changes of BP and their response to drugs have not been clearly investigated in non-human primates (NHPs) of MetS with hypertension. Here, we identified 16 elderly, hypertensive MetS rhesus monkeys from our in-house cohort. With implanted telemetry, we investigate BP changes and its circadian rhythm, together with the effect of antihypertensive drugs on BP and its diurnal fluctuation. MetS hypertensive monkeys displayed higher BP, obesity, glucose intolerance, and dyslipidemia. We also confirmed impaired 24-h BP circadian rhythm in MetS hypertensive monkeys. Importantly, Eplerenone, a mineralocorticoid receptor blocker, exerts multiple beneficial effects in MetS hypertensive monkeys, including BP reduction, 24-h BP circadian rhythm restoration, and decreased plasma concentration of inflammation factors and advanced glycation end-products. In summary, we identified a naturally-developed hypertensive MetS NHP model, which is of great value in the studies on pathogenesis of MetS-associated hypertension and development of novel therapeutic strategies. We also provided multiple novel mechanistic insights of the beneficial effect of Eplerenone on MetS with hypertension.

CaMKII is a RIP3 substrate mediating ischemia- and oxidative stress-induced myocardial necroptosis.

Regulated necrosis (necroptosis) and apoptosis are crucially involved in severe cardiac pathological conditions, including myocardial infarction, ischemia-reperfusion injury and heart failure. Whereas apoptotic signaling is well defined, the mechanisms that underlie cardiomyocyte necroptosis remain elusive. Here we show that receptor-interacting protein 3 (RIP3) triggers myocardial necroptosis, in addition to apoptosis and inflammation, through activation of Ca(2+)-calmodulin-dependent protein kinase (CaMKII) rather than through the well-established RIP3 partners RIP1 and MLKL. In mice, RIP3 deficiency or CaMKII inhibition ameliorates myocardial necroptosis and heart failure induced by ischemia-reperfusion or by doxorubicin treatment. RIP3-induced activation of CaMKII, via phosphorylation or oxidation or both, triggers opening of the mitochondrial permeability transition pore and myocardial necroptosis. These findings identify CaMKII as a new RIP3 substrate and delineate a RIP3-CaMKII-mPTP myocardial necroptosis pathway, a promising target for the treatment of ischemia- and oxidative stress-induced myocardial damage and heart failure.